Figure 1. Bulging of the branch site nucleophile is required for splicing in S. cerevisiae, but the position of the bulge within the BS-U2 snRNA duplex is flexible.

(A) Schematic of RNA-RNA interactions that contribute to the first step of splicing. The pre-mRNA is shown in black, with the branch site (BS) adenosine highlighted in blue: U2 snRNA is shown in red, U5 in grey and U6 in green. U2 positions A31 and G32 are shown in lowercase throughout for positional reference.

(B) Schematic of the BS-U2 pairing regions of wt and BS-C mutant reporters, and wt and Ψ35G U2 snRNAs used in (C).

(C) An intron BS-C mutation can be suppressed by U2 Ψ35G but not by other mutations at this position: upper panel – splicing efficiency monitored by primer extension with a 3′ exon primer on S. cerevisiae total RNA from strains carrying the indicated U2 snRNA and reporter constructs; middle panel – primer extension with an intronic primer immediately upstream of BS indicates that the Ψ35G suppressor promotes the use of a non-canonical adenosine as the branch nucleophile; lower panel – copper growth assay to verify that the mRNA observed in the upper panel is functional.

(D) Schematic of reporter and U2 constructs used in (E).

(E) Primer extensions with 3′ exon (upper panel) and BS-proximal intronic (lower panel) primers on total RNA from the indicated strains indicate that branch nucleotide bulging is required for splicing, but the position of the bulge within the BS-U2 duplex is not fixed.

(F) Schematic of reporter and U2 constructs used in (G).

(G) In vitro splicing (upper panel) and spliceosome assembly (lower panel) of the S. cerevisiae UBC4 intron carrying the indicated BS mutations, as assayed by denaturing and native PAGE, respectively. BS-C and ΔBS-A mutations allow reduced spliceosome assembly but no branching, indicating the importance of the bulged nucleotide for spliceosomal catalysis.